Massolini Gabriella, Calleri Enrica
Department of Pharmaceutical Chemistry, University of Pavia, Via Taramelli 12, 27100 Pavia, Italy.
J Sep Sci. 2005 Jan;28(1):7-21. doi: 10.1002/jssc.200401941.
The ability to rapidly and efficiently digest and identify an unknown protein is of great utility for proteome studies. Identification of proteins via peptide mapping is generally accomplished through proteolytic digestion with enzymes such as trypsin. Limitations of this approach consist in manual sample manipulation steps and extended reaction times for proteolytic digestion. The use of immobilized trypsin for cleavage of proteins is advantageous in comparison with application of its soluble form. Enzymes can be immobilized on different supports and used in flow systems such as immobilized enzyme reactors (IMERs). This review reports applications of immobilized trypsin reactors in which the IMER has been integrated into separation systems such as reversed-phase liquid chromatography or capillary electrophoresis, prior to MS analysis. Immobilization procedures including supports, mode of integration into separation systems, and methods are described.
快速高效地消化和鉴定未知蛋白质的能力在蛋白质组研究中具有重要用途。通过肽图谱鉴定蛋白质通常是通过用胰蛋白酶等酶进行蛋白水解消化来完成的。这种方法的局限性在于手动样品处理步骤以及蛋白水解消化的反应时间延长。与使用可溶性胰蛋白酶相比,使用固定化胰蛋白酶裂解蛋白质具有优势。酶可以固定在不同的载体上,并用于流动系统,如固定化酶反应器(IMER)。本综述报道了固定化胰蛋白酶反应器的应用,其中IMER在质谱分析之前已被整合到反相液相色谱或毛细管电泳等分离系统中。描述了固定化程序,包括载体、整合到分离系统中的方式以及方法。